Project Summary/Abstract Dendritic cells (DCs) play key roles in targeting immune responses to pathogens, both by functioning as antigen presenting cells and by secreting potent innate immune and inflammatory cytokines. They differentiate from hematopoietic stem cells found in the bone marrow into multiple subtypes that have more inflammatory/adaptive immune or more innate immune functions. Misregulation of these differentiation processes is the root cause of myeloproliferative disorders (MPD) including acute myeloid leukemia (AML), and the inflammatory disease Langerhans Cell Histiocytosis (LCH). These differentiation processes may be recapitulated ex vivo with key myeloid differentiation and growth factors GM-CSF and Flt3L, and prior work has shown that the NF?B signaling system is an important regulator. Yet given the complexity of this multi- transcription factor, multi-regulator signaling system, its roles in subtype-differentiation, proliferation, and in disease remain poorly understood. In the proposed project we will examine the role of NF?B control in coordinating differentiation programs of dendritic cells into two developmental pathways. Based on our preliminary results we propose the overarching hypothesis that proper stepwise assembly of the NF?B signaling system during DC differentiation pathways is critical for phasing proliferation and maturation; while classical I?B?, -?, -? mediate transient NF?B inflammatory responses, the recently described I?Bsome plays a critical buffering and coordinating role in DC differentiation. Misregulation of the I?Bsome leads to severe but surprisingly different phenotypes in the two DC developmental pathways. We will combine an experimentally validated mathematical model of NF?B signaling during dendritic cell differentiation and function, with quantitative biochemical, flow cytometric, and live cell fluorescence microscopy, interferometry and lineage tracking studies involving knockin reporter mice, and a number of novel genetic mouse strains to uncover the molecular basis of NF?B misregulation associated with myeloprolferative disorders (MPD) and Langerhans Cell Histiocytosis (LCH). We will apply these insights about the I?Bsome regulation to examine patient samples and the efficacy and risks of potential drug targets.